A reporter gene assay measures the activity of a gene's promoter, and thus the expression of the proteins encoded by the gene or genes that are under the control of the promoter. These proteins can be involved in a variety of cellular activities. Therefore, a reporter gene assay also measures cellular activities associated with the proteins. These assays generally use techniques in molecular biology to make nucleic acid constructs that place a gene under the control of a promoter. These constructs can then be stabely or transiently introduced into a cell, such as a mammalian cell (see, Gorman, C. M. et al,. Mol. Cell Biol. 2: 1044-1051 (1982); and Alam. J. and Cook, J. L., Anal.Biochem. 188: 245-254, (1990)). When the promoter is activated, the reporter gene is expressed and a reporter protein is produced. The reporter protein can be, for example, an enzyme that converts a substrate into a detectable product. The product can be measured qualitatively or quantitatively as a measure of the activation of the promoter and thus the level of activity of the genes normally under the control of that promoter.
Several reporter genes are known in the art and some are commercially available (see, Alam and Cook, supra). The reporter gene can be inserted within a plasmid that is particularly suited for an organism and molecular biology manipulations. Promoters of interest can be inserted into cloning sites so that the expression of the reporter gene is under the control of the promoter (see, Rosenthal, N., Methods Enzymol. 152: 704-720 (1987); and Shiau, A. and Smith, J. M., Gene 67: 295-299 (1988)). Known methods are used to introduce these plasmids into a cell type or whole organism (see, Sambrook et al., Molecular Biology, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); and Nolan, In: Molecular Cloning, Cold Spring Harbor Laboratory Press, (1989)). The plasmids can also encode a resistance marker, such as resistance to antibiotics so that successfully transfected cells can be selected, identified, and cultured.
Substrates for enzymatic reporter proteins can be chromogenic or fluorescent. In some assays, a fluorescent substrate changes fluorescence properties upon conversion by the reporter enzyme to a fluorescent product. Preferably, a fluorescent product is highly fluorescent and can become trapped within the cell rather than being detectable in the media surrounding the cells. These features allow the expression of the reporter protein to be monitored and measured in individual cells rather than in a population of cells (see, WO 96/30540 to Tsien, published Oct. 3, 1996).
One way to increase the sensitivity of a fluorescent reporter assay is to maximize the amount of a fluorescent signal generated by a single reporter enzyme. An optimal enzyme will convert 10.sup.5 substrate molecules per second under saturating conditions (see, Stryer, L. Introduction to enzymes. In: Biochemistry, New York: W. H. Freeman and company, 1981, pp. 103-134). Beta-lactamases can cleave about 10.sup.3 molecules of a preferred substrates per second (see, Chang, Y. H. et al., Proc.Natl.Acad.Sci.USA 87: 2823-2827 (1990)). A preferred fluorescent product can produce up to 10.sup.6. In practice, a small fraction of the photons generated by the fluorescent product will be detected.
A preferred fluorescent substrate has a high turnover and optical properties such as high extinction and high fluorescence quantum yield. This present invention provides such preferred fluorescent substrates and provides additional benefits as well.